Plants and animals use small RNAs (microRNAs [miRNAs] and siRNAs) as guides for posttranscriptional and epigenetic regulation. In plants, miRNAs and trans-acting (ta) siRNAs form through distinct biogenesis pathways, although they both interact with target transcripts and guide cleavage. An integrated approach to identify targets of Arabidopsis thaliana miRNAs and ta-siRNAs revealed several new classes of small RNA-regulated genes, including conventional genes such as Argonaute2 and an E2-ubiquitin conjugating enzyme. Surprisingly, five ta-siRNA-generating transcripts were identified as targets of miR173 or miR390. Rather than functioning as negative regulators, miR173- and miR390-guided cleavage was shown to set the 21-nucleotide phase for ta-siRNA precursor processing. These data support a model in which miRNA-guided formation of a 5' or 3' terminus within pre-ta-siRNA transcripts, followed by RDR6-dependent formation of dsRNA and Dicer-like processing, yields phased ta-siRNAs that negatively regulate other genes.

Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.

Micro-RNAs (miRNAs) are regulatory molecules that mediate effects by interacting with messenger RNA (mRNA) targets. Here we show that Arabidopsis thaliana miRNA 39 (also known as miR171), a 21-ribonucleotide species that accumulates predominantly in inflorescence tissues, is produced from an intergenic region in chromosome III and functionally interacts with mRNA targets encoding several members of the Scarecrow-like (SCL) family of putative transcription factors. miRNA 39 is complementary to an internal region of three SCL mRNAs. The interaction results in specific cleavage of target mRNA within the region of complementarity, indicating that this class of miRNA functions like small interfering RNA associated with RNA silencing to guide sequence-specific cleavage in a developmentally controlled manner.

The molecular basis for virus-induced disease in plants has been a long-standing mystery. Infection of Arabidopsis by Turnip mosaic virus (TuMV) induces a number of developmental defects in vegetative and reproductive organs. We found that these defects, many of which resemble those in miRNA-deficient dicer-like1 (dcl1) mutants, were due to the TuMV-encoded RNA-silencing suppressor, P1/HC-Pro. Suppression of RNA silencing is a counterdefensive mechanism that enables systemic infection by TuMV. The suppressor interfered with the activity of miR171 (also known as miRNA39), which directs cleavage of several mRNAs coding for Scarecrow-like transcription factors, by inhibiting miR171-guided nucleolytic function. Out of ten other mRNAs that were validated as miRNA-guided cleavage targets, eight accumulated to elevated levels in the presence of P1/HC-Pro. The basis for TuMV- and other virus-induced disease in plants may be explained, at least partly, by interference with miRNA-controlled developmental pathways that share components with the antiviral RNA-silencing pathway.

MicroRNAs (miRNAs) are approximately 21-nucleotide noncoding RNAs that regulate target transcripts in plants and animals. In addition to miRNAs, plants contain several classes of endogenous small interfering RNAs (siRNAs) involved in target gene regulation and epigenetic silencing. Small RNA libraries were constructed from wild-type Arabidopsis (Arabidopsis thaliana) and mutant plants (rdr2 and dcl3) that were genetically enriched for miRNAs, and a computational procedure was developed to identify candidate miRNAs. Thirty-eight distinct miRNAs corresponding to 22 families were represented in the libraries. Using a 5# rapid amplification of cDNA ends procedure, the transcription start sites for 63 miRNA primary transcripts from 52 MIRNA loci (99 loci tested) were mapped, revealing features consistent with an RNA polymerase II mechanism of transcription. Ten loci (19%) yielded transcripts from multiple start sites. A canonical TATA box motif was identified upstream of the major start site at 45 (86%) of the mapped MIRNA loci. The 5#-mapping data were combined with miRNA cloning and 3#-PCR data to definitively validate expression of at least 73 MIRNA genes. These data provide a molecular basis to explore regulatory mechanisms of miRNA expression in plants. MicroRNAs (miRNAs) are approximately 21-nt noncoding RNAs that posttranscriptionally regulate expression of target genes in plants and animals (Bartel, 2004). Mature miRNAs are generated through multiple processing steps from primary transcripts (pri-miRNA) that contain imperfect foldback structures. In animals, MIRNA genes are transcribed by RNA polymerase II (pol II; Bracht et al., 2004;Cai et al., 2004;Lee et al., 2004), yielding a pri-miRNA that is processed initially by the nuclear RNaseIII-like enzyme Drosha (Lee et al., 2003). The resulting premiRNA transcripts are transported to the cytoplasm and processed by Dicer to yield mature miRNAs (Lee et al., 2002). Less is known about the miRNA biogenesis pathway in plants, although most or all miRNAs require Dicer-like1 (DCL1;Park et al., 2002;Reinhart et al., 2002). The lack of a Drosha ortholog in plants and the finding that DCL1 functions at multiple steps during biogenesis of miR163 suggest that plant miRNA biogenesis may differ somewhat from animals (Kurihara and Watanabe, 2004). miRNAs in both animals and plants incorporate into an effector complex known as the RNA-induced silencing complex and guide either translation-associated repression or cleavage of target mRNAs (Bartel, 2004).Computational and molecular cloning strategies revealed nearly 100 potential MIRNA genes in the Arabidopsis (Arabidopsis thaliana) genome (Llave et al., 2002a;Mette et al., 2002;Park et al., 2002;Reinhart et al., 2002;Palatnik et al., 2003;Jones-Rhoades and Bartel, 2004;Sunkar and Zhu, 2004;Wang et al., 2004). These miRNAs target mRNAs encoding proteins that include a variety of transcription factors involved in development, miRNA/small interfering RNA (siR-NA) metabolic or effector components (DCL1, Argonaute1 [AGO1], and AGO...